Doctoral Dissertations

Date of Award

5-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

J. Erby Wilkinson

Committee Members

Stephen J. Kennel, David O. Slauson, Karla Matteson

Abstract

The scurfy (sf) mutation manifests itself as a severe, rapidly fatal lymphoproliferative disease, resulting in death in hemizygous males (sf/Y) due to a wasting syndrome by 22-26 days of age. Earlier studies of sf mice focussed on the thymus as the primary factor in the etiology and expression of the disease, and a genetically sf thymus was shown to be required for the development of scurfy. It was hypothesized that inappropriate thymic selection was the primary defect in this mutation. Later studies indicated that CD4+ T cells, matured in a sf thymus, were the crucial lymphocyte subpopulation responsible for the disease. The hypothesis was developed that failure of thymic negative selection resulted in the release of autoreactive T cells to the periphery. This hypothesis was expanded to predict a defect in the control of the peripheral immune system in sf mice. A recently-derived strain of mice which are homozygous-deficient for CTLA-4, a molecule vital for down-regulating activated T cells, develops an antigen independent disease which closely resembles scurfy. The mouse mutation Ipr (lymphoproliferation) also has biological similarities to sf and has a defect in the Fas-FasL pathway of apoptosis. A recent study indicated that in sf mice both the Fas-FasL and CD28/CTLA-4/B7.1B7.2 pathways were activated. The Fas- FasL apoptotic pathway was determined as functional. Despite activation of the CTLA- 4 pathway, sf peripheral lymphocytes showed negligible levels of apoptosis ex vivo.

It was not known if the disease-causing sf CD4+ T cells were reacting to a specific antigen, or if the lymphoproliferation typically seen in scurfy was spontaneous. To determine if the lymphoproliferation was antigen dependent, and if that antigen were exogenous or endogenous, sf was bred onto an H-2 compatible, BALB/c transgenic line (DO 11.10) in which 75-95% of the T cells express receptors (TCRs) for a chicken ovalbumin peptide (OVA 323-339). Thus sf/Y OVA mice should have at least 75% of their T cells reactive specifically with OVA alone. This allowed for experimental manipulation of CD4-I- T cells by administration of OVA peptide, both in vivo and in vitro. sf/Y OVA mice had prolonged lifespans (up to 6 months) and less severe scurfy-related abnormalities as compared to both the original sf strain and nontransgenic sf/Y wt littermates. sf/Y OVA mice successfully deleted self-reactive T cells when challenged in vivo with OVA peptide, indicating that a genetically sf thymus was capable of central deletion. In vitro, sf/Y OVA thymic and splenic T cells showed greater proliferation when incubated with low concentrations of OVA peptide as compared to the splenic and thymic T cells from wt/Y OVA and non-transgenic counterparts, indicating an alteration in sf/Y OVA T cell sensitivity to antigen.

sf/Y OVA mice did eventually develop scurfy-like disease and die with manifestations similar to those of the original sf strain, most likely due to the remaining, non-OVA TCR-bearing cells (5-25% of the total T cell population). To eliminate this possibility, the Ragl knock-out mouse was chosen for breeding to the sf/Y OVA progeny to deplete the normal T and B cell populations. The combination of Ragl -/-, transgenic OVA TCRs (either homozygous or heterozygous), and sf produce mice with virtually all of their mature T cells reactive strictly to OVA peptide. This was confirmed by flow cytometry and a DO 11.10-specific antibody, KJ1 -26. None of these mice developed the scurfy disease over two years of observation.

Histopathology confirmed the lack of lymphoproliferation in ail organs. Experiments on RaglKO/sf/Y OVA in vivo showed that these mice were able to thymically delete self-reactive T cells. When challenged in vivo with OVA over a period of several weeks, mice did not develop any scurfy clinical signs, although their harvested thymic and splenic T cells showed evidence of activation. Splenic RaglKO/sf/Y OVA cells incubated in vitro with OVA had an alteration in T cell antigenic response. At the highest concentrations of peptide tested, RaglKO/sf/Y OVA splenic cells proliferated significantly less than RaglKO/wt OVA controls. However, and most significantly, RaglKO/sf/Y OVA peripheral T cells did not properly cease proliferation in response to high doses of OVA peptide, a process referred to as tolerization, which is signalled intracellularly via the CTLA-4 pathway and is vital to the peripheral control of potentially self-reactive T cells. When primed in vivo, then challenged ex vivo with OVA peptide, RaglKO/wt OVA mice tolerized, RaglKO/sf/Y OVA mice did not, even when given twice the dose of peptide.

These findings indicated that mice with CD4+ T cells incapable of responding to non-OVA antigens did not develop scurfy, consistent with the hypothesis that the sf mutation results in an exuberant immune response that is otherwise normal. Peripheral sf lymphocytes responded to antigen differently in vitro than controls, indicating an alteration in stimulating and tolerizing thresholds. Based on these observations, the sf mutation produces an antigen-dependent disease which is likely the result of T cells with altered sensitivities to antigenic stimulation and tolerization.

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